Our goal is to explore strategies aimed at understanding and overcoming T cell tolerance as a way to improve adoptive immunotherapy for cancer and chronic viral infections. CD8+ T lymphocytes represent a branch of the immune system responsible for recognizing and destroying cancerous or infected cells. Adoptive T cell immunotherapy strives to harness this ability for the treatment of human disease by transferring large numbers of reactive T cells into patients. However, the anticipated clinical benefits of this treatment have not been realized for a majority of patients, highlighting the need for novel therapeutic approaches. Several obstacles to success have been identified including deletion and induction of tolerance in transferred T cells, as tumor antigens are often aberrantly or over-expressed self-antigens protected by mechanisms of peripheral self- tolerance. The tumor microenvironment also promotes the induction of tolerance by failing to provide appropriate co-stimulatory signals to T cells. Additionally, tolerance can be induced during chronic viral infections due to persistent over-stimulation (exhaustion), leading to expression of negative regulatory molecules that impair T cell receptor (TCR) signaling. Thus, while essential for protection against autoimmunity, tolerance represents a substantial challenge to mounting an effective CD8+ T cell immune response. Despite advances in T cell biology and tumor immunology, the molecular mechanisms that regulate tolerance remain largely unknown, hampering efforts to overcome in vivo tolerance for therapeutic applications. Recently, induced expression of genetically engineered TCR (dual-TCR) on transferred T cells has gained enthusiasm as a means to direct immune responses toward malignant or infected cells. These dual-TCR T cells have shown promise not only as cellular reagents for therapy, but also as tools to more effectively interrogate the mechanisms responsible for tolerance induction, which is regulated at least in part by proximal defects at independently functioning TCR complexes rather than global cellular defects. Experiments designed to understand at the molecular level how dually expressed TCR influence one another and direct T cell activity may provide key insight into how enduring therapeutic responses might be achieved in patients. The long-term objectives of this research are to evaluate strategies that exploit dual-TCR expression as means to rescue T cell tolerance, enhance the efficacy and durability of adoptively transferred T cells against established diseases, and to utilize such cells to explore the intracellular events that control induction of T cell tolerance. The specific aims are (1) to rescue tumor-reactive CD8+ T cells tolerized during adoptive immunotherapy; (2) to elucidate the molecular mechanisms that regulate induction of CD8+ T cell tolerance; and (3) to restore function of exhausted CD8+ T cells during chronic viral infection. These experiments are designed to examine new avenues of immunotherapy and provide mechanistic insight into T cell tolerance and rescue such that these approaches may be translated to a broad range of human malignancies and infections.